ISOTOPIC DIFFERENTIATION OF INTERACTIONS AS RANDOM OR TARGETED (I-DIRT)

这个子项目是利用资源的许多研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 通过亲和标记的蛋白质分离蛋白质复合物为研究生物系统提供了一个强大的工具，但该技术往往受到非特异性相互作用蛋白质的共富集的影响。我们开发了一种新技术（I-DIRT），在免疫纯化中区分污染物与真正的相互作用物，克服了定义蛋白质复合物中最具挑战性的问题。该技术的基本原理是将含有亲和标记蛋白质的同位素轻细胞与不含标记蛋白质的同位素重细胞混合。 在亲和标记的蛋白质复合物的分离和共富集蛋白质的胰蛋白酶消化后，特异性相互作用蛋白质通过质谱法鉴定为同位素轻的，而非特异性相互作用蛋白质表现为同位素轻和重的混合物。 我们已经在J. Proteome Res.（2005）4，1752-6中发表了这项工作，并且还发表了这项工作的应用（例如，D.J. Lee，S.J.W.巴斯比，L.F. Westblade，B.T.大肠杆菌O 157：H7 Sakai RNA聚合酶复合物的Chait免疫分离和I-DIRT质谱分析。 J. Bacteriology 190（2008）1284-1289）。 我们将继续研究这种技术的更精细的细节，重点是评估具有相对较高的解离率的特定相互作用物的效果。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of non-specifically interacting proteins. We developed a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. The basic principle of this technique is mixing isotopically light cells that contain an affinity-tagged protein with isotopically heavy cells that do not contain a tagged protein. After isolation of the affinity-tagged protein complex and trypsin digestion of the co-enriching proteins, specifically interacting proteins are identified by mass spectrometry as isotopically light, whilst non-specifically interacting proteins appear as a mixture of isotopically light and heavy. We have published this work in J. Proteome Res. (2005) 4, 1752-6 and have also published applications of this work (e.g., D.J. Lee, S.J.W. Busby, L.F. Westblade, B.T. Chait Immunoisolation and I-DIRT mass spectrometric analysis of the Escherichia coli O157:H7 Sakai RNA polymerase complex. J. Bacteriology 190 (2008) 1284-1289). We continue to investigate the finer details of this technique, with an emphasis on evaluating the effect of specific interactors that have relatively high offrates.